Diagnostic kit

ABSTRACT

Compounds and a method for imaging myocardial perfusion, including administering to a patient a compound linked to an imaging moiety, wherein the compound binds MC-1, and scanning the patient using diagnostic imaging. Kits including the compound or precursor compounds linked or not linked to an imaging moiety are also described.

FIELD OF INVENTION

The present disclosure is directed to imaging agents, pharmaceuticalcompositions and methods for imaging myocardial perfusion, comprisingadministering to a patient a compound linked to an imaging moiety,wherein said compound binds MC-1, and scanning the patient usingdiagnostic imaging. The invention also relates to kits comprising saidimaging agent or precursor compounds linked or not linked to an imagingmoiety.

BACKGROUND OF INVENTION

Coronary artery disease (CAD) is a leading cause of death in the Westernworld. Imaging techniques for diagnosis and prognosis are very importantfor the treatment of CAD to reduce the mortality. Imaging for evaluationthe myocardial blood flow to determine the treatment necessary (oftensurgery) is a critical part of CAD healthcare. Currently Single PhotonEmission Computer Tomography (SPECT) is the mainstay of CAD imaging butimproved diagnostic methods are needed.

Heart cells, myocardia, have a very high intracellular density, weightpercentage, of mitochondria. It was therefore reasoned that compoundsthat selectively bind to mitochondria would be enriched in myocardia.Certain insecticides act through binding to the mitochondria complex I(MCI). Included in this group of insecticides are rotenone, pyridaben,tebufenpyrad and fenazaquin. It was believed that such compoundsselective for MCI could be used for imaging mitochondrial rich tissue. Apatent for the use of labelled rotenone for myocardial blood flowimaging was disclosed in 2001.

In 2005 BMS filed a patent (WO 2005/079391), describing ¹⁸F labelledcompounds based on the insecticides pyridaben, tebufenpyrad andfenazaquin for the use as PET-ligands for the diagnosis and imaging ofmycocardial blood flow in CAD. The patents from BMS were later acquiredby Lantheus Medical imaging. One of the compounds based on pyridaben,flurpiradaz (BMS747158), has been extensively studied and is now inphase III studies for myocardial imaging. Flurpiridaz has been found toprovide superior assessment of myocardial function than the SPECT agent99mTc sestamibi.

Respiratorius, a pharmaceutical company based in Lund, Sweden, has beenworking on discovering novel bronchodilating drugs. A central part ofRespiratorius' discovery work is screening small molecules that canrelax human airway tissue ex vivo. During this process a series of novel1,8 naphthyridines were discovered as potent bronchorelaxing compounds(described in patent application WO/2010/097410). Upon furtherpharmacological studies it was found that members of this class ofcompounds bound to and inhibited mitochondrial complex I.

SUMMARY OF THE INVENTION

It has surprisingly been found that a bronchodilating compound belongingto a class of 1,8-naphthyridines also can inhibit mitochondrial functionby relaxing the airway smooth muscle, alter mitochondrial function orbind to mitochondrial complex I. If the compounds are labelled with animaging moiety a valuable diagnostic marker for myocardial perfusionimaging will be available.

The invention relates to an imaging agent having the structure

wherein R₁ is H, F, CF₃, Cl, R is a linker and X is an imaging moiety

or an analogue or pharmaceutically salt of said imaging agent.

In a second aspect the invention relates to a pharmaceutical compositioncomprising the imaging agent shown above and a pharmaceuticallyacceptable carrier, diluent, buffer. The imaging agent and compositiongives rise to a high cardiac uptake to non-target ratio with minimalredistribution. It will also result in better image quality and diseasedetection and diagnosis. An almost linear myocardial uptake versus flow:up to 5 mL/min/g (high first-pass extraction) will be obtained. Itallows quantification of absolute myocardial flow and will be effectivewith both exercise and pharmacologic stress. It will have an appropriatesafety profile and be available as unit dose (such as 18F-labeledcompound).

In a third aspect the invention relates to a method of imaging a heartin a patient comprising: administering to the patient a diagnosticallyeffective amount of the imaging agent or pharmaceutical compositiondefined above, and obtaining an image of the heart of the patient. In afinal aspect the invention relates to a diagnostic kit comprising acompound having the following formula

wherein R₁ is H, F, CF₃, Cl, R is a linker and X is a leaving groupselected from the group consisting of tosylate, mesylate, triflate,nonaflate and halogen or an analogue of said compound and wherein saidkit can be used to prepare an imaging agent as defined above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a synthetic pathway how to produce an imaging compound.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the context of the present application and invention, the followingdefinitions apply:

The term “Pharmaceutically acceptable salt” refers to those salts whichretain the biological effectiveness and properties of the free bases andwhich are obtained by reaction with inorganic or organic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid,succinic acid, tartaric acid, citric acid, and the like.

An analogue is a molecule that differs in chemical structure from aparent compound, for example a homolog (differing by an increment in thechemical structure, such as a difference in the length of an alkylchain), a molecular fragment, a structure that differs by one or morefunctional groups, a change in ionization. Structural analogues areoften found using quantitative structure activity relationships (QSAR),with techniques such as those disclosed in Remington (The Science andPractice of Pharmacology, 19^(th) Edition (1995), chapter 28).

The term “linking group,” as used herein, refers to a portion of amolecule that serves as a spacer between two other portions of themolecule. Linking groups may also serve other functions as describedherein. Examples of linking groups include linear, branched, or cyclicalkyl, aryl, ether, polyhydroxy, polyether, polyamine, heterocyclic,aromatic, hydrazide, peptide, peptoid, or other physiologicallycompatible covalent linkages or combinations thereof.

In a first embodiment the invention relates to an imaging agent havingthe structure

wherein R₁ is H, F, CF₃, Cl, R is a linker and X is an imaging moiety oran analogue or pharmaceutically acceptable salt of said imaging agent.

R may be a straight alkyl, ethyleneglycol (ether) or polyethylenglycol.

One example being

wherein R is a linker and X is an imaging moiety.

Another example being an imaging agent with the formula shown below:

wherein X is an imaging moiety.

X may be a halogen isotope, such as a fluorine, bromine, chlorine oriodine isotope. Examples includes ¹⁸F, ¹⁹F_(,) ¹²⁰I, ¹²¹I, ¹²²I, ¹²³I,¹²⁴I, ¹²⁵I, ¹²⁷I, ¹³¹I, ³⁵Cl, ³⁷Cl, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁹Br, ⁸⁰Br,^(80m)Br, ⁸¹Br or ⁶⁴Cu. In a specific example ¹⁸F or ¹⁹F is used.

In another embodiment the invention relates to a pharmaceuticalcomposition comprising the imaging agent as defined above and apharmaceutically acceptable carrier, diluent, or buffer.

“Pharmaceutically acceptable” means a non-toxic material that does notdecrease the effectiveness of the biological activity of the activeingredients, i.e., the peptide(s), polypeptide(s) or variants thereof.Such pharmaceutically acceptable buffers, carriers or excipients arewell-known in the art (see Remington's Pharmaceutical Sciences, 18thedition, A. R Gennaro, Ed., Mack Publishing Company (1990) and handbookof Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., PharmaceuticalPress (2000).

The term “buffer” is intended to mean an aqueous solution containing anacid-base mixture with the purpose of stabilising pH.

The term “diluent” is intended to mean an aqueous or non-aqueoussolution with the purpose of diluting the peptide in the pharmaceuticalpreparation. The diluent may be one or more of saline, water, humanserum albumin, e.g., tris (hydroxymethyl) aminomethane (and its salts),phosphate, citrate, bicarbonate, alcohols including ethanol, sterilewater, physiological saline, or balanced ionic solutions containingchloride and or bicarbonate salts or normal blood plasma cations such ascalcium, potassium, sodium and magnesium. The labelled compound may bepresent in from 1.0 to 50 millicuries, such as 1.0-10, 10-20, 20-30,30-40, 40-50 millicuries

The pharmaceutical formulations according to the invention may beadministered systemically. Routes of administration include parenteral(intravenous, subcutaneous, and intramuscular), oral, parenteral,vaginal and rectal. Suitable preparation forms are, for exampledispersions, suspensions, aerosols, droples or injectable solution inampule form and also preparations with protracted release of activecompounds, in whose preparation excipients, diluents or carriers arecustomarily used as described above.

The imaging agents of the present invention may be used in methods ofimaging, including methods of imaging in a patient. For example, themethod may comprise administering the imaging agent to the patient byinjection (e.g., intravenous injection), infusion, or any other knownmethod, and imaging the heart of the subject wherein an event ofinterest is located.

The useful dosage to be administered and the particular mode ofadministration will vary depending upon such factors as age, weight, thediagnostic use contemplated, and the form of the formulation, forexample, suspension, emulsion, microsphere, liposome, or the like, aswill be readily apparent to those of ordinary skill in the art.

Typically, dosage is administered at lower levels and increased untilthe desirable diagnostic effect (e.g., production of an image) isachieved. In one embodiment, the above-described imaging agents may beadministered by intravenous injection, usually in saline solution, at adose of about 0.1 to about 100 mCi per 70 kg body weight (and allcombinations and subcombinations of dosage ranges and specific dosagestherein), or, in some embodiments, at a dose of about 0.5 to about 50mCi. Imaging is performed using techniques well known to the ordinarilyskilled artisan.

Another aspect of the present invention provides diagnostic kits for thepreparation of imaging/diagnostic agents for determining (e.g.,detecting), imaging, and/or monitoring at least a portion of the heart.Diagnostic kits of the present invention may comprise one or more vialscontaining a sterile, non-pyrogenic, formulation comprising apredetermined amount of a reagent (e.g., contrast agent precursor) ofthe present invention, and optionally other components such as chelatingagents, solvents, buffers, neutrlization aids, lyophilization aids,stabilization aids, solubilization aids and bacteriostats, as describedmore fully below.

Some non-limiting examples of buffers useful in the preparation ofcontrast agents and kits include, for example, phosphate, citrate,sulfosalicylate, and acetate buffers. A more complete list can be foundin the United States Pharmacopoeia.

Some non-limiting examples of lyophilization aids useful in thepreparation of contrast agents and kits include, for example, mannitol,lactose, sorbitol, dextran, FICOLL.®. polymer, and polyvinylpyrrolidine(PVP).

Some non-limiting examples of stabilization aids useful in thepreparation of contrast agents and kits include, for example, ethanol,ascorbic acid, ethanol, cysteine, monothioglycerol, sodium bisulfite,sodium metabisulfite, gentisic acid, and inositol.

Some non-limiting examples of solubilization aids useful in thepreparation of contrast agents and kits include, for example, ethanol,glycerin, polyethylene glycol, propylene glycol, polyoxyethylenesorbitan monooleate, sorbitan monoloeate, polysorbates,poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) block copolymers(“Pluronics.®.”) and lecithin.

Some non-limiting examples of bacteriostats useful in the preparation ofcontrast agents and kits include, for example, benzyl alcohol,benzalkonium chloride, chlorobutanol, and methyl, propyl, or butylparaben.

The compounds and compositions according to the invention may be usedwith imaging techniques such as positron emission tomography (PET) andSingle Photon Emission Computed Tomography (SPECT). PET imaging is adiagnostic examination that involves the acquisition of physiologicimages based on the detection of radiation from the emission ofpositrons from a radionuclide compound administered to the patient. Theradionuclide compound is typically administered via intravenousinjection. Different colours or degrees of brightness on a PET imagerepresent different levels of tissue or organ function. SPECT imaging isa three-dimensional technique combined with computer assistedreconstruction of images of organs to reveal both anatomy and function.As with PET imaging, patients undergoing SPECT imaging is administered aradioactive tracer. PET and SPECT images may be used to evaluate avariety of diseases, and are commonly used in the fields of oncology,cardiology, and neurology.

Methods of Synthesizing Contrast Agents

Typically, imaging agents described herein may be synthesized byreacting at least a first component and a second component, such that abond is formed there between. For example, 18F labeled compounds may besynthesized by reacting two components via displacement of anappropriate leaving group associated with at least one component.Examples of such leaving groups include sulfonic acid esters such astoluenesulfonate (tosylate, TsO-), methanesulfonate (mesylate, MsO-), ortrifluoromethanesulfonate (triflate, TfO-), nonaflate or halogen. Theleaving group may also be a halide, a phosphineoxide (via Mitsunobureaction), or an internal leaving group (such as an epoxide or cyclicsulfate). Purification is generally performed via salt removal byreverse-phase chromatography.

Representative methods of making the compounds are described in thefollowing examples. The foregoing chemical transformations may beconducted using techniques which would be readily apparent to one ofordinary skill in the art, in combination with the teachings describedherein. In some cases, methods of synthesizing the contrast agents mayinclude the use of one or more reaction solvents. Representativereaction solvents include, for example, DMF, NMP, DMSO, THF, ethylacetate, dichloromethane, and chloroform. The reaction solution may bekept neutral or basic by the addition of an amine such as triethylamineor DIEA. In some cases, the chemical transformations (e.g., reactions)may be carried out at ambient temperatures and protected from oxygen andwater with a nitrogen, argon or helium atmosphere.

In some embodiments, temporary protecting groups may be used to preventother reactive functionality, such as amines, thiols, alcohols, phenols,and carboxylic acids, from participating or interfering in the reaction.Representative amine protecting groups include, for example,tert-butoxycarbonyl and trityl (removed under mild acidic conditions),Fmoc (removed by the use of secondary amines such as piperidine), andbenzyloxycarbonyl (removed by strong acid or by catalytichydrogenolysis). The trityl group may also used for the protection ofthiols, phenols, and alcohols. In certain embodiments the carboxylicacid protecting groups include, for example, tert-butyl ester (removedby mild acid), benzyl ester (usually removed by catalytichydrogenolysis), and alkyl esters such as methyl or ethyl (usuallyremoved by mild base). All protecting groups may be removed at theconclusion of synthesis using the conditions described above for theindividual protecting groups, and the final product may be purified bytechniques which would be readily apparent to one of ordinary skill inthe art, in combination with the teachings described herein.

Following examples are intended to illustrate, but not to limit, theinvention in any manner, shape, or form, either explicitly orimplicitly.

EXAMPLES Example 1 Synthesis of an Imaging Compound Example 1

N-[[3-fluoro-4-(2-fluoroethoxymethyl)phenyl]methyl]-2-methyl-1,8-naphthyridine-3-carboxamide

A flask with a solution of 19 mg2-[[2-fluoro-4-[[(2-methyl-1,8-naphthyridine-3-carbonyl)amino]methyl]phenyl]methoxy]ethyl4-methylbenzenesulfonate (0.036 mmol), 26 mg Kryptofix 222(4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane) (0.069mmol) and 4 mg KF (0.069 mmol) in 1.0 ml dry MeCN was added to apreheated oil bath and heated at 90 C for 30 min. The reaction mixturewas cooled to room temperature and diluted with water. The mixture wasextracted twice with EtOAc. The combined organic phases were washed withbrine, dried (MgSO4) and concentrated. Flash chromatography gave 9.6 mg(72%).

¹H NMR (CDCl₃) δ 8.97 (dd, 1H), 8.03 (s,1H), 8.01 (m, 1H), 7.42 (m, 2H),7.19 (dd, 1H), 7.13 (m, 1H), 7.08 (t, 1H), 4.67 (m, 2H), 4.65 (s, 3H),4.53 (m, 1 H), 3.80 (m, 1 H), 3.73 (m, 1 H).

2-[[2-fluoro-4-[[(2-methyl-1,8-naphthyridine-3-carbonyl)amino]methyl]phenyl]methoxy]ethyl4-methylbenzenesulfonate

25 mg tosylchloride (0.13 mmol) was added to a solution of 40 mgN-[[3-fluoro-4-(2-hydroxyethoxymethyl)phenyl]methyl]-2-methyl-1,8-naphthyridine-3-carboxamide(0.11 mmol), 23 μl diisopropylethylamine (6.13 mmol) and 13 mg DMAP(0.11 mmol) in 1.0 ml CH2Cl2 at room temperature. The solution wasstirred for 2 h. The reaction mixture was placed directly on a SiO2column and purified by flash chromatography (CH2Cl2/MeOH 50:1). Gave 52mg (90%).

¹H NMR (CDCl₃) δ 8.89 (m, 1H), 7.99 (s, 1H), 7.82 (m, 1H), 7.73 (m, 3H),7.31 (m, 4H), 7.13 (m, 2H), 4.65 (d, 2H), 4.51 (s, 2H), 4.15 (d, 2H),3.68 (m, 2H), 2.77 (s, 3H), 2.41 (s, 3H)

N-[[3-fluoro-4-(2-hydroxyethoxymethyl)phenyl]methyl]-2-methyl-1,8-naphthyridine-3-carboxamide

45 μl oxalyl chloride (0.53 mmol) was added to a mixture of 50 mg2-methyl-1,8-naphthyridine-3-carboxylic acid (0.27 mmol) in 3 ml CH2Cl2with one drop DMF. The reaction mixture was stirred for 1.5 h and thenevaporated to dryness under reduced pressure. The residue was dissolvedin 3 ml CH2Cl2. 4 mg DMAP (0.03 mmol) and 188 μl triethyl amine (1.35mmol) were added to the solution followed by 54 mg2-[[4-(aminomethyl)-2-fluoro-phenyl]methoxy]ethanol (0.27 mmol). Thereaction mixture was stirred for 4 h and then diluted with water. Thephases were separated and the aqueous phase was extracted with CH2Cl2.The combined organic phases were dried (MgSO4) and concentrated. Flashchromatography (SiO2, CH2Cl2/MeOH 20:1) gave 36 mg (36%) of the titlecompound.

¹H NMR (CDCl₃) δ 8.82 (m, 1H), 7.96 (s, 1H), 7.95 (m, 1H), 7.88 (m, 1H),7.31 (m, 2H), 7.10 (m, 2H), 4.60 (d, 2H), 4.56 (s, 2H), 3.75 (m, 2H),3.61 (m, 2H), 2.68 (s, 3H)

1. A kit comprising: an imaging agent having the structure

wherein R₁ is H, F, CF₃, Cl, R is a linker and X is an imaging moiety orradioactive isotope or an analogue or pharmaceutically acceptable saltof said imaging agent; and an instruction how to use said kit.
 2. Adiagnostic kit comprising: a compound having the following formula

where R₁ is H, F, CF₃, Cl, R is a linker and X is a leaving groupselected from the group consisting of tosylate, mesylate, triflate,nonaflate and halogen or an analogue of said; wherein said kit can beused to prepare an imaging agent having the structure

wherein R₁ is H, F, CF₃, Cl, R is a linker and X is an imaging moiety orradioactive isotope or an analogue or pharmaceutically acceptable saltof said imaging agent.
 3. A kit comprising: a pharmaceutical compositioncomprising: an imaging agent having the structure

wherein R₁ is H, F, CF₃, Cl, R is a linker and X is an imaging moiety orradioactive isotope or an analogue or pharmaceutically acceptable saltof said imaging agent; a pharmaceutically acceptable carrier, diluent,buffer; and an instruction how to use said kit.